4.4. Western Blot Analysis

Western blots were carried out according to methods described in [45,46]. In short, five gerbils/group were deeply anesthetized by an intraperitoneal injection of 90 mg/kg pentobarbital sodium (JW pharm. Co., Ltd., Seoul, Korea) [47] at each point in time after ischemia. Under the anesthesia, their brains were removed, and the tissues of CA1 and CA2/3 were respectively dissected using surgical blades. The obtained tissues were homogenized with 0.05 M PBS (pH 7.4) containing 0.1 mM ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) (pH 8.0), 10 mM ethylenediaminetetraacetic acid (EDTA, pH 8.0), 0.2% Nonidet P-40, 100 mM β-glycerophosphate, 15 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 50 mM NaF, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM dithiothreitol (DTT). The homogenized CA1 and CA2/3 tissues were centrifuged, and their supernatants were taken for assessing protein level using a Micro BCA kit with bovine serum albumin (Pierce Chemical, Rockford, IL, USA) as the standard. The aliquots including total protein (20 μg) were boiled in loading buffer consisting of 150 mM Tris (pH 6.8), 0.3% bromophenol blue, 3 mM DTT, 6% SDS, and 30% glycerol. Thereafter, the samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the gels were transferred to nitrocellulose membranes from Pall Company (East Hills, NY, USA) at 350 mA under 4 °C for 1.5 h. In this experiment, to avoid non-specific staining, the nitrocellulose membranes were immersed into 5% defatted milk at 23 °C for 1 h. Next, those membranes were immunoreacted with each primary antibody at 4 °C for 8 h: (1) rabbit anti-HO-1 (diluted 1:2000; Abcam, Cambridge, UK) and (2) rabbit anti-β-actin (diluted 1:2000; Sigma-Aldrich, St. Louis, MO, USA). In succession, the membranes were incubated with HRP-conjugated donkey anti-rabbit IgG (diluted 1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 1 h. Finally, a luminol-based chemiluminescence kit from Pierce (Thermo Fisher Scientific Inc., MA, USA) was used for enhancement of visualization.